Inhibition Of HBV Replication And Growth Ability Of Hepatocelluar Carcinoma Cells Study By HBS RNAi

RNA interference provides the mechanism by which specific post-transcriptional silencing of gene expression can occur. Small interfering RNA (siRNA) has been used repeatedly to down-regulate viral gene expression and inhibit viral replication in mammalian cells. RNAi might be an efficient antiviral therapy for some obstinate illness. Hepatitis B virus is a member of the hepadnaviridae family. Human hepatitis B virus is a small-enveloped DNA virus which can cause acute and chronic infection of the liver and lead to hepatocellular carcinoma. Chronic hepatitis B virus infection is a major health problem, especially in Asia, and patients with chronic hepatitis are at increased risk of developing hepatocellular carcinoma. Hepetocellularcarcinoma (HCC) is one of the rare human neoplasms associated with viral infections. Hepatitis B virus infection is a major cause of hepatocellular carcinoma in Southeast Asia including China. The risk of HCC development is greatly increased in chronic viral carriers exposed to other recognized risk factors. The role of HBV in tumour formation appears to be complex and may involve both direct and indirect mechanisms. Prolonged expression of the viral regulatory protein HBx and the large envelope protein (LHBs) may contribute in deregulating the cellular transcription program and proliferation control, and sensitize liver cells to carcinogenic factors. Chronic active hepatitis is recognized as an important risk factor for hepatocellular carcinoma. Recent data demonstrate that serum HBV DNA level is a strong risk predictor of HCC independent of HBeAg, serum alanine aminotransferase level and liver cirrhosis. Thus, HBV should be considered as having synergistic effects with chronic inflammation.The planning of targeted sequence is the key step of RNAi experiment. There are five methods in the execution of RNAi in current research. The chemically synthesized small interference RNA (siRNA) can effectively decrease the expression of targeted gene, but the expensive cost restricts its widely application. Vector mediated RNAi is suitable for the research after specific sequences are screened and identified, while the procedure of screening is comparatively complicated. Cock-tail and siRNA expression frame methods can't be applied in the screening of targeted sequence. Generally speaking, most specific targeted sequence in RNAi is selected in the coding region of mRNA, either located in the 5'end, 3'end or intermedial sequence. The selection in the non-coding region of mRNA (5'UTR or 3'UTR) was recently reported. Several recent reports have demonstrated that the cellular expression of siRNA or shRNA targeting HBs gene inhibits viral replication. These studies have involved the introduction of synthetic siRNA or the genetic transfer of DNA expression cassettes capable of producing siRNA/shRNA in hepatoma cells. However, these reports did not refer to HBV-related HCC. Another potential approach for delivering siRNA and inhibiting HBV could involve the use of the structure of pre-microRNAs (pre-miRNAs). siRNA encoded by miRNA structure has significant superiority over by above means. No reports, to our knowledge, have silenced HBs gene by siRNAs incorporated into miRNA backbone, especially a lentiviral system regulated by CMV promoter.Among the four proteins that are encoded by the HBV genome, LHBs and HBV x protein(HBx) are most potentially oncogenic factors. It is known that LHBs plays an important role in hepatocarcinogenesis, but the exact signal transduction way and molecular mechanisms of LHBs in HCC are not well understood. The expression of HBs in the majority of HCC samples strongly suggests that it may play a critical role in regulating the proto-oncoproteins necessary for tumorigeniciy and the anti-apoptotic ability of HCC. To understand this further, we employed RNAi and used miRNA-based lentiviral vector to knock down the expression of HBs in HBV-producing cell lines, and further addressed the oncological gene expression changes.We applied the RNA Structure software to predict the second structure of mRNA of HBs, and selected three pieces of sequence according to the planning principle, which fell into the intermedial region. The siRNA sequence was within conservative regions from different HBV genotypes, including B and C. By use of pSuper vecter we produced the siRNA of HBs and transfected it combined with HBV plasmid into human embryonic kidney 293T cells. The interference effect of these two targeted sequences was identified by real-time PCR and ELISA analysis, which showing one of the sequences (seq. 2) can silence the mRNA expression by 80%, the other sequences do not demonstrate any effect. In order to further study HBs function in hepatoma cell line, we cloned the DNA sequence of seq. 2 into lentiviral vector, being packaged by lentivirus to be used in subsequent experiments. Two oligonucleotides were annealed and introduced into EcoRⅠ/XbaⅠsites of plasmid PGCLM-001. Lentiviruses were generated by cotrasfecting lentiviral vector and packaging vectors in 293T cells by using lipofectamine 2000. Supernatants were collected 48 h after transfection, filtered through a 0.45μm membrane. Herein, a lentiviral microRNA-based RNAi system was developed to drive hepatitis B virus x antigen–specific short miRNA in HepG2.2.15 cells. HBsAg in the supernatant of the cells were analyzed by ELISA, and intracellular protein was extracted and the HBsAg was measured by western blot. Total cellular RNA was isolated HepG2.2.15 cells with Trizol reagent according to the manufacturer's protocol. cDNA was synthesized by RT-PCR and HBs mRNA levels was analyzed by realtime PCR. The cell-free supernatant HBV DNA levels were analyzed by fluorogenic quantitative polymerase chain reaction. Three×107 HepG2.2.15 cells were injected subcutaneously into both flanks of BALB/c nu/nu male mice of 3-4 weeks of age. The tumor sizes were measured once every 5-10 days. The HBs-related oncological genes were analyzed by microarray analysis.The inhibitory effect was efficient. Decreases in the corresponding viral transcript levels were observed. The inhibitory effect was observed within 96 h and was still evident 9 days after the initial treatment with RNAi. Secreted HBsAg was reduced by >70% in cell culture, and intracellular HBsAg was significantly reduced too. The RNAi caused a significant inhibition in the levels of viral mRNA relative to the controls. A significant reduction in virion production was also observed for the 2.2.15 cells. RNAi targeting HBs in HepG2.2.15 cells domonstrated significant reduction in tumor development in nude mice.Our findings thus indicate that lentiviral microRNA-based RNAi can specifically mediate the down-regulation of viral gene expression leading to a reduction in viron production. Lentiviral micro-RNA based RNAi may be useful for therapy in HBV and hepatocellular carcinoma. HBs plays an important role in tumorigenicity in HCC. HBV infection might trigger specific oncogenic pathways, thus playing a role beyond stimulation of host immune responses and chronic necro-inflammatroy liver disease.